An antibody is an immunoglobulin molecule that has a specific amino acid residue sequence and thus binds only with the antigen that induced its synthesis (its immunogen) or with a closely related antigen (or immunogen). Immunoglobulin molecules include two kinds of polypeptide chains.
Each molecule consists of larger identical polypeptide chains referred to as heavy chains (H chains) and two identical smaller chains referred to as light chains (L chains). These polypeptide chains are held together by disulfide bonds and by noncovalent bonds, which are primarily hydrophobic. The heavy and light polypeptide chains are synthesized in vivo on separate ribosomes, assembled in the cell, and are secreted as an intact immunoglobulin molecule.
The understanding of the structure and function of immunoglobulins has been facilitated by studies of fragments produced by enzymatic cleavage of the antibody molecule. For example, treatment of an antibody molecule with the enzyme papain produces two antigen-binding fragments (designated "Fab") and a complement-binding fragment (designated "Fc"), which contains no antigen-binding capability but determines important biological characteristics of the intact immunoglobulin molecule. Treatment of an antibody molecule with the enzyme pepsin, on the other hand, produces a single antigen-binding fragment [designated "F(ab').sub.2 "] and a complement-binding fragment (also designated "Fc") that is somewhat smaller than the Fc fragment produced by papain.
The constant region of H chains permits the differentiation of immunoglobulins into classes and subclasses, and confers certain biological properties such as the ability to activate complement, to cross the placenta, and to bind to polymorphonuclear leukocytes or macrophages.
In particular, five immunoglobulin classes (IgG, IgA, IgM, IgD, and IgE) are recognized on the basis of structural differences of their heavy chains including the amino acid residue sequence and length of the polypeptide chain. The antigenic determinants on the heavy chains also permit the identification and quantitation of the immunoglobulin classes by immunochemical assay techniques.
The amino-terminal one-half of the light chains and the amino-terminal one-quarter of the heavy chains of an immunoglobulin molecule vary in their amino acid residue sequence and are termed the variable regions (V regions) of the polypeptide chains. Portions of the V region of one heavy and of one light polypeptide chain constitute the site for antigen binding. A considerable variation in the amino acid residue sequence of the variable region of an immunoglobulin molecule can exist which produces the many different antibody specificities. A region of extreme variability in the primary sequence within a variable region is called a hypervariable region. [Capra et al., Proc. Natl. Acad. Sci. USA, 71, 845 (1974).]
Hypervariable regions contain residues that contact the antigen and bind to it on the basis of mutual complementarity (complementarity-determining region or CDR). The regions are discontinuous at the level of primary structure but converge at the level of tertiary structure to form the continuous, highly contorted sequence of the binding site.
The specificity of the molecular binding site of an antibody is termed its idiotype. The term idiotype denotes the unique variable (V) region sequences produced by each type of antibody-forming cell. An antibody having a binding site specificity for the binding site of another antibody is termed an anti-idiotypic antibody.
The same amino acid sequence variation that produces the antigen binding specificity of an immunoglobulin also determines which idiotypic determinants are present. Thus, particular idiotypes are almost invariably associated with immunoglobulins of a particular specificity. As such, idiotypes can serve as antigenic markers for immunoglobulins with a particular specificity and, by virtue of their surface immunoglobulin, B lymphocytes of the same specificity.
The terms "cross-reactive" and "cross-reactivity" refer to the ability of an antibody to bind antigens other than its idio-specific antigen. Cross-reactive anti-idiotypic antibodies can be divided into two major groups.
One group comprises those anti-idiotypic antibodies that recognize idiotypic antigenic determinants that are associated with specific amino acid residue sequences in the heavy and light chain variable regions. Anti-idiotype antibodies of this group often reflect the action of inherited immunoglobulin structural genes. Consequently, these antibodies do not cross-react in subjects that are not genetically similar.
The second group includes anti-idiotypic antibodies that are cross-reactive to the internal image of the antigen. This type of anti-idiotypic antibody is elicited by immunization with an intact immunoglobulin and usually recognizes idiotypic antigenic determinants as a result of a particular quaternary interaction of the light and heavy chains. The antigenic site recognized by this group of anti-idiotypic antibodies, however, is not associated with a particular light or heavy chain amino acid residue sequence.
Because the antibody binding site bears the internal image of the antigen; i.e., mimics the size, shape, charge and/or van der Waals attraction of the antigen, the second group of anti-idiotypic antibody binds to many different antibodies of the same specificity. The idiotypes recognized by such antibodies can be produced by individuals with different genetic backgrounds and are controlled by genes that bear no special relationship.
Anti-idiotype immunotherapy can be very useful in the treatment of autoimmune disease, by neutralizing pathological auto-antibodies. Anti-idiotypic therapy can be highly specific. But such therapy suffers from the disadvantages associated with passive administration since the anti-idiotypic antibodies must be produced in a non-human species. Therefore, a significant possibility exists that an individual so treated will develop an immune response against the passively administered antibodies, which response can negate any potential therapeutic effect. This is particularly true because the antibodies must be administered many times to produce the desired result.
Moreover, all anti-idiotypic antibodies have previously been generated by immunizing the host with the target immunoglobulin. The resulting polyclonal antisera must then be extensively purified to produce antibodies having the desired anti-idiotypic specificity. The selected, purified, "monoclonal" antibodies must then be carefully tested to determine their specificities.
The structural correlates of idiotypes have been sought in several well-defined antibody systems. See Kunkel et al., Science, 140, 1218 (1963); Capra et al., Proc. Natl. Acad. Sci. USA, 71, 4032 (1974); Weigert et al., J. Exp. Med., 139, 137 (1974); Klapper et al., Ann. Immunol. (Inst. Pasteur), 127C, 261 (1976); Schilling et al., Nature, 283, 35 (1980); Capra et al., Immunol. Today, 3, 332 (1982); and Capra et al., Immunol. Today, 4, 177 (1983). These studies suggest that a hypervariable region (containing a complementarity-determining region or a CDR) of an immunoglobulin is the structural correlate of an idiotypic determinant.
In particular, in the murine anti-dextran system, one private (or individual) idiotype and one public (or cross-reactive) idiotype were assigned to the third and second hypervariable regions, respectively, of the heavy chain. Schilling et al., supra. However, in most systems, it has proven extremely difficult to associate a particular idiotypic determinant with a specific amino acid residue sequence (Capra et al., Immunol. Today, 4, supra.) Rather, anti-idiotypic antibodies elicited by immunization with an intact immunoglobulin usually recognize determinants dependent upon a particular quaternary interaction or "internal image" of both of the light and heavy chains. (Capra et al., Id.)
Lerner et al. have been successful in obtaining protection of animals by the use of vaccines against pathogens that utilize synthetic polypeptides having amino acid residue sequences of short to moderate length as immunogens. See Sutcliffe et al., Science, 219, 660 (1983). Such synthetic polypeptides induce antibodies specific for predefined determinants of intact proteins.
As described herein, synthetic polypeptide technology can avoid the previously described difficulties associated with conventional anti-idiotypic therapy. According to the present invention, described in detail hereinafter, polypeptides having relatively short amino acid residue sequences that substantially correspond to the portion of the immunoglobulin primary sequence that forms the idiotype can be synthesized, coupled to an appropriate carrier and inoculated into animal hosts, including humans, as immunogens to raise antibodies. The resulting antisera recognize the synthetic polypeptide having an amino acid residue which immunologically corresponds substantially to a primary amino acid residue sequence of a portion of an immunoglobulin variable region, including an idiotypic antigenic determinant. The antisera are therefore idiotype specific. Such antisera produced by synthetic polypeptides are thus of predetermined specificity and the necessity for extensive purification and specificity testing is eliminated substantially.
Briefly, the polypeptide alone is not immunogenic in most cases. Small molecules such as the peptides of this invention, can be coupled to appropriate antigenic carriers to form conjugates. The resulting peptide-carrier conjugate is immunogenic. Antibodies are produced to both the peptide antigen and antigens of the carrier. However, anti-carrier antibodies so produced do not interfere with the specificity of diagnostic assays, effective immunoregulation or any use contemplated by the invention.
In addition, such synthetic polypeptides alone, as conjugates or as polymers can be administered to individuals to raise antibodies that immunoreact with the particular idiotypes of that individual. Autologous anti-idiotypic antibodies are well documented and are widely believed to be very important in immunoregulation. One advantage in the use of synthetic polypeptide-containing antigens (immunogens) is that antibodies reactive with otherwise non-immunogenic determinants can be elicited. Therefore, appropriate synthetic polypeptides can induce anti-idiotypic antibodies in an individual that are directed against a particular idiotype of that individual whereas this could not be achieved by immunizing with the intact immunoglobulin which results in antibodies against substantially all the antigenic determinants of the immunoglobulin.
Thus, an individual can be actively immunized against a pathological idiotype and the number of therapeutic interventions required can be substantially reduced compared to conventional immunization with an intact immunoglobulin. Also, the possibility of an immune response against the anti-idiotypic antibodies can be reduced substantially as compared to antibodies produced in another animal species and passively administered to a human.
Polypeptides can also be synthesized to mimic an antigen under attack by pathological auto-antibodies. These polypeptides can block or inhibit the interaction between the antigen and the undesirable auto-antibodies, thereby significantly impeding the disease process.
It is believed that certain idiotypes occur very frequently in particular syndromes. Synthetic polypeptides, corresponding to such idiotypes can be used to elicit antibodies of predetermined specificity for such syndromes, and can then be applied in the diagnosis and treatment of that syndrome.